Presence of an O-glycosidically linked hexasaccharide in fetuin

Examination by gel filtration, thin layer and anion exchange chromatography of the O-linked carbohydrate units released from fetuin by alkaline borohydride treatment indicated the presence in this glycoprotein of an acidic glucosamine-containing hexasaccharide in addition to the previously described tetra- and trisaccharides. The structure of the hexasaccharide was determined to be NeuAc alpha 2----3Gal beta 1----3[NeuAc alpha 2----3Gal beta 1----4GlNAc beta 1----6]GalNAc, on the basis of exoglycosidase digestion, periodate oxidation, and methylation analysis as well as hydrazine-nitrous acid fragmentation. The latter procedure when carried out on the reduced asialohexasaccharide yielded Gal----2-deoxygalactitol and Gal----anhydromannose which were shown to be derived, respectively, from Gal----N-acetylgalactosaminitol and Gal----GlcNAc sequences. Reductive amination of the Gal----anhydromannose disaccharide with [14C] methylamine permitted identification of its linkage as 1----4. While Diplococcus pneumoniae endo-alpha-DN-acetylgalactosaminidase acting on asialofetuin released the sialic acid-free tetra- and trisaccharides (Gal beta 1----3GalNAc), this enzyme did not cleave the peptide attachment of the asialohexasaccharide (Gal beta 1----3 [Gal beta 1----4GlcNAc beta 1----6] GalNAc). The number of O-linked hexa-, tetra-, and trisaccharides per fetuin molecule was determined to be 0.2, 0.7, and 2.1, respectively, on the basis of galactosaminitol analyses. The absence of O-linked N-acetylglucosamine-containing tetra- or pentasaccharides in fetuin suggest that the attachment of this sugar is a rate-limiting step; furthermore, the limited occurrence of the hexasaccharide may indicate that the addition of sialic acid to Gal beta 1----3GalNAc to form the NeuAc alpha 2----3Gal linkage precludes action of the GlcNAc transferase to form the branch point on the GalNAc residue.     

Evidence for an O-glycan sialylation system in brain. Characterization of a beta-galactoside alpha 2,3-sialyltransferase from rat brain regulating the expression of an alpha-N-acetylgalactosaminide alpha 2,6-sialyltransferase activity

We present evidence for the existence in rat brain of several sialyltransferases able to sialylate sequentially asialofetuin. [14C]Sialylated glycans of asialofetuin were analyzed by gel filtration. Three types of [14C]sialylated glycans were synthesized: N-glycans and monosialylated and disialylated O-glycans. The varying effects of N-ethylmaleimide, lysophosphatidylcholine (lysoPtdCho) and trypsin, were helpful in the identification of these different sialyltransferases. One of them, selectively inhibited by N-ethylmaleimide, was identified as the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase previously described [Baubichon-Cortay, H., Serres-Guillaumond, M., Louisot, P. and Broquet, P. (1986) Carbohydr. Res. 149, 209-223]. This enzyme was responsible for the synthesis of disialylated O-glycans. LysoPtdCho and trypsin selectively inhibited the enzyme responsible for the synthesis of monosialylated O-glycan. N-ethylmaleimide, lysoPtdCho and trypsin did not inhibit Neu5Ac transfer onto N-glycans, giving evidence for three different molecular species. To identify the enzyme responsible for monosialylated O-glycan synthesis, we used another substrate: Gal beta 1----3GalNAc--protein obtained after galactosylation of desialylated ovine mucin by a GalNAc-R:beta 1----3 galactosyltransferase from porcine submaxillary gland. This acceptor was devoid of N-glycans and of NeuAc in alpha 2----3 linkages on the galactose residue. When using N-ethylmaleimide we obtained the synthesis of only one product, a monosialylated structure. After structural analysis by HPLC on SAX and SiNH2 columns, we identified this product as Neu5Ac alpha 2----3Gal beta 1----3GalNAc. The enzyme leading to synthesis of this monosialylated O-glycan was identified as a Gal beta 1----3GalNAc-R:alpha 2----3 sialyltransferase. When using lysoPtdCho and trypsin, sialylation was completely abolished, although the Neu5Ac alpha 2----3Gal beta 1----3GalNAc-R:alpha 2----6 sialyltransferase was not inhibited. We provided thus evidence for the interpendence between the two enzymes, the alpha 2----3 sialyltransferase regulates the alpha 2----6 sialyltransferase activity since it synthesizes the alpha 2----6 sialyltransferase substrate.     

Asparagine-linked sugar chains of fetuin: occurrence of tetrasialyl triantennary sugar chains containing the Gal beta 1----3GlcNAc sequence

Asparagine-linked sugar chains were quantitatively released from fetuin by hydrazinolysis. Structural analysis of the sugar chains by sequential exoglycosidase digestion in combination with methylation analysis and Smith degradation revealed that most of them have typical biantennary (8%) and triantennary (74%) structures containing different amounts of N-acetylneuraminic acid residues. In addition, an unusual tetrasialyl triantennary sugar chain (17%) containing the Gal beta 1----3GlcNAc sequence in the outer chain moiety was detected, and its structure was elucidated as NeuAc alpha 2----3Gal beta 1----3(NeuAc alpha 2----6)-GlcNAc beta 1----4(NeuAc alpha 2----6Gal beta 1----4GlcNAc beta 1----2)Man alpha 1----3(NeuAc alpha 2----3Gal beta 1----4GlcNAc beta 1----2Man alpha 1----6)Man beta 1----4GlcNAc beta 1----4GlcNAc.     

Identification of a alpha-NeuAc-(2----3)-beta-D-galactopyranosyl N-acetyl-beta-D-galactosaminyltransferase in human kidney

Microsomal preparations from human kidney were found to contain enzymic activity capable to transfer N-acetylgalactosamine from UDP-N-acetylgalactosamine to native bovine fetuin. The acceptor structures on the fetuin molecules were identified as N- as well as O-linked glycans with a markedly higher incorporation into the N-linked carbohydrate chains. Analysis of the alkali-labile transferase products by thin-layer chromatography indicated that the enzyme is able to synthesize structures having mobilities identical with those found on glycophorin from Cad erythrocytes. Mild acid treatment and enzymic hydrolysis with N-acetylhexosaminidase from jack beans of the N-linked transferase products suggested that beta-D-GalpNAc-(1----4)-[alpha-NeuAc-(2----3)]-beta-D-Galp-(1----s tructures were formed by the enzymic reaction on both N- and O-linked acceptors. The enzyme might, therefore, be involved in the biosynthesis of Sda (and Cad) antigenic structures. By use of various oligosaccharides, glycopeptides, and glycolipids having well characterized carbohydrate sequences, the acceptor-substrate specificity of the N-acetylgalactosaminyltransferase was determined. The enzyme generally recognized alpha-NeuAc-(2----3)-beta-D-Gal groups as acceptors, but in a certain conformation. Thus, tri- and tetra-saccharide alditols, native human glycophorin A, and GM3 were not acceptor substrates although they carry the potential disaccharide acceptor unit. When these structures were presented as sialyl-(2----3)-lactose or as a tryptic peptide from glycophorin A, they were shown to be rather good acceptor substrates for the N-acetyl-beta-D-galactosaminyltransferase from human kidney.     

Structures of asparagine-linked oligosaccharides of the glycoprotein fetuin having sialic acid linked to N-acetylglucosamine

In the accompanying paper (Bendiak et al., 1989), the separation of a series of oligosaccharides released from asparagine residues of fetuin was described. A series of NMR experiments, which included one- and two-dimensional nuclear Overhauser enhancement, two-dimensional correlation spectroscopy, and two-dimensional relayed-coherence spectroscopy, as well as permethylation analyses, established a Gal beta 1----3(NeuAc alpha 2----6)GlcNAc beta 1----4Man unit common to a series of purified structures. These oligosaccharides contained either three, four, or five glycosidically linked sialic acid residues. The NeuAc residue in alpha 2----6 linkage to GlcNAc gives rise to diagnostic chemical shift perturbations of particular proton signals in the oligosaccharides.     

Conformational analysis of the tumor-associated carbohydrate antigen 19-9 and its Lea blood group antigen component as related to the specificity of monoclonal antibody CO19-9

A structural comparison between the synthetic, tumor-associated 19-9 tetrasaccharide, NeuAc alpha 2----3Gal beta 1----3GlcNAc(4----1 alpha Fuc)-O(CH2)8CO2CH3 and its Lea blood group antigen component, Gal beta 1----3GlcNAc(4----1 alpha Fuc)-O(CH2)8CO2CH3 was carried out by two-dimensional 1H NMR spectroscopy and hard-sphere energy calculations. Significant chemical shift differences between the two molecules were detected only for protons at or near the linkage site of NeuAc to the Lea trisaccharide core. Coupling constants for the ring protons of both molecules did not suggest major deviation from the 4C1 chair conformation for Gal and GlcNAc, the 1C4 conformation for Fuc, or the 2C5 conformation for NeuAc. Two-dimensional nuclear Overhauser enhancement experiments revealed through-space, inter-proton interactions that corresponded to some extent with those predicted by diffraction data and hard-sphere energy minimization programs for both saccharides. However, a significant number of interactions did not obey the distance dependence predicted from a rigid structure model. These data suggest that, while the average conformation of the 19-9 antigen's Lea core may be invariant to NeuAc alpha 2----3Gal linkage, the dynamics of the Lea trisaccharide are altered upon sialylation. Data also indicate that the terminal NeuAc linkage is more flexible than the inter-residue bonds of the core trisacharide. This analysis, in combination with the fact that the monoclonal anti-19-9 antibody CO 19-9 does not cross-react with the Lea antigen, provides evidence in favor of NeuAc as an epitope-creating unit involved directly at the antibody binding site. However, given the possible role of variable dynamics in epitope formation, these results do not preclude crucial roles in antibody recognition for regions on the 19-9 antigen that are distanced from NeuAc.     

Metabolism of asialoglycoproteins in cultured rat hepatocytes: evidence for receptor mediated uptake and degradation which is not feed-back regulated

1. Cultured rat hepatocytes took up and degraded asialofetuin by a saturable mechanism. 2. The uptake was specific for desialylated fetuin; native fetuin and was taken up to a much lesser extent. 3. Degradation was inhibited by lysosomotropic agents; both uptake and degradation were, however, unaffected by cycloheximide. 4. The uptake was also unaffected by incubating the cells for 24 hr in the presence of large extracellular concentrations of unlabelled asialofetuin. 5. The data suggest that the asialoglycoprotein receptor is recycled and not subject to down-regulation.     

Biosynthesis of the sialyl-Lex determinant carried by type 2 chain glycosphingolipids (IV3NeuAcIII3FucnLc4, VI3NeuAcV3FucnLc6, and VI3NeuAcIII3V3Fuc2nLc6) in human lung carcinoma PC9 cells

The pathway for synthesis of three glycosphingolipids bearing a common sialyl-Lex determinant (NeuAc alpha 2----3Gal beta 1----4[Fuc alpha 1----3]GlcNac beta 1----R) from their type 2 lactoseries precursors has been studied using the 0.2% Triton X-100-soluble fraction from human lung carcinoma PC9 cells. Two enzymes were found to be required for their synthesis: (i) an alpha 1----3 fucosyltransferase, the properties of which have been characterized as being similar to the enzyme from human small cell lung carcinoma NCI-H69 cells (Holmes, E. H., Ostrander, G. K., and Hakomori, S. (1985) J. Biol. Chem. 260, 7619-7627); and (ii) an alpha 2----3 sialyltransferase that was efficiently solubilized by 0.2% Triton X-100 and required divalent metal ions and 0.3% Triton CF-54 for optimal activity at pH 5.9 in cacodylate buffer. Biosynthesis of the sialyl-Lex determinant was shown to proceed via sialylation of nLc6 and nLc4, followed by alpha 1----3 fucosylation at the penultimate GlcNAc residues, based on the following: (i) transfer of NeuAc by PC9 cell sialyltransferase was found only when the nonfucosylated acceptors nLc4 and nLc6 were added, and none of the glycolipids with Lex structure (III3FucnLc4; V3FucnLc6; III3V3Fuc2nLc6) were sialylated; and (ii) the PC9 cell fucosyltransferase was active with both neutral and ganglioside neolacto (type 2 chain) acceptors. Transfer of fucose to VI3NeuAcnLc6 yielded mono- and difucosyl derivatives, whereas only a monofucosyl derivative was obtained when VI6NeuAcnLc6 was the acceptor. This is most probably due to different conformations at the terminus of the two acceptor gangliosides. The fucosyltransferase was incapable of transferring fucose to sialyl 2----3 lactotetraosylceramide (IV3NeuAcLc4).     

Characterization and partial purification of beta-N-acetylgalactosaminyltransferase from urine of Sd(a+) individuals

Urine from Sd(a+) individuals was found to contain a beta-N-acetylgalactosaminyltransferase that transfers N-acetylgalactosamine (GalNAc) from UDP-GalNAc to 3'-sialyllactose and glycoproteins carrying the terminal NeuAc alpha-3Gal beta group. This enzyme has been purified 174-fold by affinity chromatography on Blue Sepharose and DEAE-Sephacel chromatography in a yield of 33%. Neither endogenous incorporation nor sugar nucleotide degrading enzymes were found in the purified preparation. The transferase had a pH optimum of pH 7.5 and a requirement for Mn2+ but not for detergents. The Km for UDP-GalNAc was 66 X 10(-6) M, using fetuin as an acceptor. Like beta-GalNAc-transferase from other sources the urinary enzyme had a strict requirement for sialylated acceptors. On the basis of enzymatic and chemical treatment of the product obtained by the transfer of [3H]GalNAc to 3'-sialyllactose, we propose that the enzyme attaches GalNAc in beta-anomeric configuration to O-4 of the galactose residue that is substituted at O-3 by sialic acid. A preparation of Tamm-Horsfall glycoprotein from a Sd(a-) donor lacking beta-GalNAc was found to be the best acceptor among the glycoproteins tested. Studies on the transferase activity toward fetuin, human chorionic gonadotropin, and glycophorin A indicated that the enzyme preferentially adds the sugar to the sialylated terminal end of N-linked oligosaccharides. Unlike the beta-GalNAc-transferase bound to human kidney microsomes (F. Piller et al. (1986) Carbohydr. Res. 149, 171-184) the urinary transferase is able to transfer beta-GalNAc to the NeuAc alpha-3Gal beta-3(NeuAc alpha-6)GalNAc chains bound to the native glycophorin.     

Immunohistochemical demonstration of glycoconjugates bearing the type 2 chain-backbone structure in human fetal, normal and neoplastic gastrointestinal tract.

Immunohistochemical distributions of carbohydrate antigens based on the type 2 chain in normal as well as fetal and neoplastic tissues of human gastrointestinal tract were investigated with a monoclonal antibody (MAb) H11 (specific for type 2 chain) alone and in combination with the two MAbs MSG15 (for alpha 2----6 sialylated type 2 chain) and IB9 (for the alpha 2----6 sialylated type 2 chain and glycoproteins having NeuAc alpha 2----6Gal-NAc), and 188C1 (for short- and long-chain Lex antigens) and FH2 (for the long-chain Lex antigen). In the pyloric mucosa of secretors, the type 2 chain is oncodevelopmentally expressed, but in non-secretors it is detected in surface mucous cells of normal gastric mucosa. The alpha 2----6 sialylation, which is confined to endocrine cells of normal pyloric mucosa, occurs in fetal and carcinoma tissues. Irrespective of the secretor status, the short- and the long-chain Lex antigens can be detected in mature and immature glandular mucous cells of normal gastric mucosa, respectively; both antigens are also expressed in fetal and carcinoma tissues. In the colon, the type 2 chain and its alpha 2----6 sialylated counterpart are expressed in an oncodevelopmental manner. The short- and the long-chain Lex antigens are significantly enhanced in colonic carcinoma. The glycoproteins with NeuAc alpha 2----6GalNAc residues appear in gastric and colonic carcinoma as well as intestinalized gastric mucosa and transitional mucosa. Thus, some of these antigens were distinctively expressed in certain epithelial cells lining the normal gastrointestinal tract depending on maturation and patients' secretor status, and some were oncodevelopmental or carcinoma-associated antigens of the human gastrointestinal tract.     

Mammalian sialyltransferase ST3Gal-II: its exchange sialylation catalytic properties allow labeling of sialyl residues in mucin-type sialylated glycoproteins and specific gangliosides

While glycosyltransferases are known to display unidirectional enzymatic activity, recent studies suggest that some can also catalyze readily reversible reactions. Recently, we found that mammalian sialyltransferase ST3Gal-II can catalyze the formation of CMP-NeuAc from 5'-CMP in the presence of a donor containing the NeuAcα2,3Galβ1,3GalNAc unit [Chandrasekaran, E. V., et al. (2008) Biochemistry 47, 320-330]. This study shows by using [9-(3)H]- or [(14)C]sialyl mucin core 2 compounds that ST3Gal-II exchanges sialyl residues between CMP-NeuAc and the NeuAcα2,3Galβ1,3GalNAc unit and also radiolabels sialyl residues in gangliosides GD1a and GT1b, but not GM1. Exchange sialylation proceeds with relative ease, which is evident from the following. (a) Radiolabeleling of fetuin was ~2-fold stronger than that of asialo fetuin when CMP- [9-(3)H]NeuAc was generated in situ from 5'-CMP and [9-(3)H]NeuAcα2,3Galβ1,3GalNAcβ1,3Galα-O-Me by ST3Gal-II. (b) ST3Gal-II exchanged radiolabels between [(14)C]sialyl fetuin and [9-(3)H]NeuAcα2,3Galβ1,3GalNAcβ1,3Galα-O-Me by generating CMP-[(14)C]- and -[9-(3)H]NeuAc through 5'-CMP; only 20.3% (14)C and 28.0% (3)H remained with the parent compounds after the sialyl exchange. The [9-(3)H]sialyl-tagged MN glycophorin A, human chorionic gonadotropin β subunit, GlyCAM-1, CD43, fetuin, porcine Cowper's gland mucin, bovine casein macroglycopeptide, human placental glycoproteins, and haptoglobin were analyzed by using Pronase digestion, mild alkaline borohydride treatment, Biogel P6, lectin agarose, and silica gel thin layer chromatography. Sulfated and sialylated O-glycans were found in GlyCAM-1 and human placental glycoproteins. This technique has the potential to serve as an important tool as it provides a natural tag for the chemical and functional characterization of O-glycan-bearing glycoproteins.     

Different reactivity of two Brain sialyltransferases towards sulfhydryl reagents. Evidence for a thiol group involved in the nucleotide-sugar binding site of the NeuAcα2-3Galβ1-3GalNAc α(2–6)sialyltransferase

We have studied the amino-acid residues involved in the catalytic activity of two distinct brain sialyltransferases acting on fetuin and asialofetuin. These two enzymes were strongly inhibited byN-bromosuccinimide, a specific blocking reagent for tryptophan residues. This result suggests the involvement of such residues in the catalytic process of the two sialytransferases. Furthermore, chemical modifications by various sulfhydryl reagents led to a strong inhibition of the fetuin sialyltransferase while the asialofetuin sialyltransferase was only slightly inhibited. For a more thorough understanding of the thiol inactivation mechanism of the fetuin sialyltransferase, we studied in more detail the reactivity of this enzyme with NEM (N-ethylmaleimide), an irreversible reagent. The time-dependent inactivation followed first-order kinetics and these kinetic data afforded presumptive evidence for the binding of 1 mol NEM per mol of enzyme. Only CMP-NeuAc protected the enzyme against NEM inactivation effectively. MnCl₂ did not enhance the protective effect of CMP-NeuAc. The modifications of the fetuin sialyltransferase kinetic parameters by NEM showed a competitive mechanism between NEM and CMP-NeuAc. The results suggest the involvement of a sulfhydryl residue in or near the nucleotide-sugar binding may induce a change in conformation of the protein, leading to a decreased accessibility of this thiol group located near the nucleotide-sugar binding site). This SH group, is essential to the enzyme activity, which is not the case for the asialofetuin sialyltransferase.Abbreviations p-CMB p-chloromercuribenzoic acid - CPDS 6,6-dithiodinicotinic acid carboxypyridine disulfide - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - NEM N-ethylmaleimide - DTT dithiothreitol - Mes 2-(N-morpholino)ethane sulfonic acid - NeuAc N-acetylneuraminic acid     

Endogenous glycosphingolipid acceptor specificity of sialosyltransferase systems in intact Golgi membranes, synaptosomes, and synaptic plasma membranes from rat brain

Preparations highly enriched in Golgi complex membranes, synaptosomes, and synaptic plasma membranes (SPM) by marker enzyme analysis and electron microscopic morphology were made from the brains of 28-day-old rats. These were incubated with cytidine 5'-monophosphate-N-acetyl[14C]neuraminic acid (CMP-NeuAc) in a physiologic buffer, without detergents. Glycolipid sialosyltransferase activities (SATs) were measured by analyzing incorporation of radiolabeled NeuAc into endogenous membrane gangliosides. Golgi SAT was diversified in producing all the various molecular species of labeled gangliosides [2.64 pmol of NeuAc transferred (mg of protein)-1 h-1]. Synaptosomal SAT exhibited a lower activity [0.66 pmol (mg of protein)-1 h-1], but it was highly specific in its labeling pattern, with a marked preference for labeling NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1 Cer (GD3 ganglioside). SPM prepared from the synaptosomes retained the GD3-related SAT (or SAT-2), and the total specific activity increased [1.41 pmol (mg of protein)-1 h-1], which suggests that the location of the synaptosomal activity is in the SPM. These results indicate that SAT activity in Golgi membranes differs from that in synaptosomes with regard to endogenous acceptor substrate specificity and SAT activity of synaptosomes should be located in the synaptosomal plasma membrane. This SAT could function as an ectoenzyme in concert with ecto-sialidase to modulate the GD3 and other ganglioside population in situ at the SPM of the central nervous system.     

Evidence for a sialic acid salvaging pathway in lepidopteran insect cells

We previously described a transgenic insect cell line, Sfbeta4GalT/ST6, that expresses mammalian beta-1,4-galactosyltransferase and alpha2,6-sialyltransferase genes and produces glycoproteins with terminally sialylated N-glycans. The ability of these cells to produce sialylated N-glycans was surprising because insect cells contain only small amounts of sialic acid and no detectable CMP-sialic acid. Thus, it was of interest to investigate potential sources of sialic acids for sialoglycoprotein synthesis by these cells. We found that Sfbeta4GalT/ST6 cells can produce sialylated N-glycans when cultured in the presence but not in the absence of fetal bovine serum. The serum component(s) supporting N-glycan sialylation by Sfbeta4GalT/ST6 cells is relatively large-it was not removed by dialysis in a 50,000-molecular-weight cutoff membrane. Serum-free media supplemented with purified fetuin but not asialofetuin supported N-glycan sialylation by Sfbeta4GalT/ST6 cells. The terminally sialylated N-glycans isolated from fetuin also supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells. Finally, serum-free medium supplemented with N-acetylneuraminic acid or N-acetylmannosamine supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells but to a much lower degree than serum or fetuin. These results provide the first evidence of a sialic acid salvaging pathway in insect cells, which begins to explain how Sfbeta4GalT/ST6 and other transgenic insect cell lines can sialylate recombinant glycoproteins in the absence of a more obvious source of CMP-sialic acid.     

A monoclonal antibody that recognizes a cluster of a disaccharide, NeuAc alpha(2----6)GalNAc, in mucin-type glycoproteins

The structure of an epitopic carbohydrate recognized by a monoclonal antibody, MLS 102, was determined. A disaccharide, NeuAc alpha (2----6)GalNAc, the major prosthetic group of ovine submaxillary mucin (OSM) and related synthetic glycosides, NeuAc alpha(2----6)GalNAc alpha----Ser, NeuAc alpha(2----6)GalNAc beta----Ser, and NeuAc alpha (2----6)GalNAc beta----propyl, reacted with MLS 102 to similar extents, but the reaction was considerably weaker compared to that of OSM. This difference in reactivity could be ascribed to the occurrence of a cluster of the disaccharide on OSM. Purification of MLS 102-reactive antigens from a Triton X-100 extract of LS 180 cells by means of immunoaffinity chromatography gave mucin fractions (cMLS 102 antigen) with an OSM-like domain. Correlation between the content of the disaccharide, NeuAc alpha(2----6)GalNAc, in mucins and their reactivity with MLS 102 was observed.     

Biosynthesis of the O-glycosidically linked oligosaccharide chains of fetuin. Indications for an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase with a narrow acceptor specificity in fetal calf liver

Fetal calf liver microsomes were found to be capable of sialylating 14C-galactosylated ovine submaxillary asialomucin. The main oligosaccharide product chain could be obtained by beta-elimination under reductive conditions and was identified as NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAcol (where GalNAcol represents N-acetylgalactosaminitol) by means of high performance liquid chromatography (HPLC) analysis and methylation. The branched trisaccharide Gal beta 1 leads to 3(NeuAc alpha 2 leads to 6)-GalNAcol and the disaccharide NeuAc alpha 2 leads to 6GalNAcol were not formed. Very similar results were obtained when asialofetuin and antifreeze glycoprotein were used as an acceptor. When 3H-sialylated antifreeze glycoprotein ([3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc-protein) was incubated with fetal calf liver microsomes and CMP-[14C]NeuAc, a reduced tetrasaccharide could be isolated. The structure of this product chain appeared to be [3H]NeuAc alpha 2 leads to 3Gal beta 1 leads to 3([14C]NeuAc alpha 2 leads to 6)GalNAcol, as established by means of HPLC analysis, specific enzymatic degradation with Newcastle disease virus neuraminidase, and periodate oxidation. These data indicate that fetal calf liver contains two sialyltransferases involved in the biosynthesis of the O-linked bisialotetrasaccharide chain. The first enzyme is a beta-galactoside alpha 2 leads to 3 sialyltransferase which converts Gal beta 1 leads to 3 GalNAc chains to the substrate for the second enzyme, a (NeuAc alpha 2 leads to 3Gal beta 1 leads to 3)GalNAc-protein alpha 2 leads to 6 sialyltransferase. The latter enzyme does not sialylate GalNAc or Gal beta 1 leads to 3GalNAc units but is capable of transferring sialic acid to C-6 of GalNAc in NeuAc alpha 2 leads to 3Gal beta 1 leads to 3GalNAc trisaccharide side chains, thereby dictating a strictly ordered sequence of sialylation of the Gal beta 1 leads to 3 GalNAc units in fetal calf liver.     

The c-series gangliosides GT3, GT2 and GP1c are formed in rat liver Golgi by the same set of glycosyltransferases that catalyse the biosynthesis of asialo-, a- and b-series gangliosides.

Biosynthesis of the c-series gangliosides GT3, GT2 and GP1c was studied in Golgi derived from rat liver. Competition experiments show that the synthesis of ganglioside GT2 (GalNAc beta 1----4-(NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal- beta 1----4Glc beta 1----1Cer) from GT3 (NeuAc alpha 2----8NeuAc alpha 2----8-NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) seems to be catalysed by the same N-acetylgalactosaminyl-transferase (GalNAc-T), which converts GM3 (NeuAc alpha 2----3Gal beta 1----4Glc beta 1----1Cer) to GM2 (GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1Cer). Similar competition experiments suggest moreover that the sialytransferase V (SAT V), which catalyses the synthesis of GT1a (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4- (NeuAc alpha 2----3)-Gal beta 1----4Glc beta 1----1Cer) from GD1a (NeuAc alpha-2----3Gal beta 1----3GalNAc beta 1----4(NeuAc alpha 2----3)Gal beta 1----4Glc beta 1----1-Cer) appears to be identical to the enzyme that catalyses the synthesis of GP1c (NeuAc alpha 2----8NeuAc alpha 2----3Gal beta 1----3-GalNAc beta 1----4(NeuAc alpha 2----8-NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta-1----4Glc beta 1----4Glc beta 1----1Cer) from GQ1c (NeuAc alpha 2----3Gal beta 1----3Gal-NAc beta 1----4 (NeuAc alpha 2----8NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4-Glc beta 1----1Cer).(ABSTRACT TRUNCATED AT 250 WORDS)     

The structures of N- and O-glycosidic carbohydrate chains of a chondroitin sulfate proteoglycan isolated from the media of the human aorta

A large Mr chondroitin sulfate proteoglycan was extracted from the media of human aorta under dissociative conditions and purified by density-gradient centrifugation, ion-exchange chromatography, and gel filtration chromatography. Removal of a contaminating dermatan sulfate proteoglycan was accomplished by reduction, alkylation and rechromatography on the gel filtration column. After chondroitinase ABC treatment, the proteoglycan core was separated from a residual heparan sulfate proteoglycan by a third gel filtration chromatography step. As assessed by radioimmunoassay, the isolated proteoglycan core was free of link protein, but possessed epitopes that were recognized by antisera against the hyaluronic acid binding region of bovine cartilage proteoglycan as well as those that were weakly recognized by anti-keratan sulfate antisera. Following beta-elimination of the protein core, the liberated low Mr oligosaccharides were partially resolved by Sephadex G-50 chromatography, and their primary structure was determined by 500-MHz1H NMR spectroscopy in combination with compositional sugar analysis. The N-glycosidic carbohydrate chains, which were obtained as glycopeptides, were all biantennary glycans containing NeuAc and Fuc; microheterogeneity in the NeuAc----Gal linkage was detected in one of the branches. The N-glycosidic glycans have the following overall structure: (Formula: see text). The majority of the O-glycosidic carbohydrate chains bound to the protein core were found to be of the mucin type. They were obtained as glycopeptides and oligosaccharide alditols, and possessed the following structures: NeuAc alpha(2----3)Gal beta(1----3)GalNAc-ol, [NeuAc alpha(2----3)Gal beta(1----3)[NeuAc alpha(2----6)]GalNAc-ol, and NeuAc alpha-(2----3) Gal beta(1----3)[NeuAc alpha(2----3)Gal beta(1----4)GlcNAc beta(1----6)] GalNAc-ol. The remainder of the O-glycosidic carbohydrate chains bound to the isolated proteoglycan were the hexasaccharide link regions of the chondroitin sulfate chains that remained after chondroitinase ABC treatment of the native molecule. These latter glycans, which were obtained as oligosaccharide alditols, had the following structure (with GalNAc free of sulfate or containing sulfate bound at either C-4 or C-6): delta 4,5GlcUA beta(1----3)GalNAc beta(1----4)GlcUA beta(1----3)Gal beta(1----3)Gal beta(1----4)Xyl-ol.     

Structural and conformational analysis of sialyloligosaccharides using carbon-13 nuclear magnetic resonance spectroscopy

The analysis of the carbon-13 chemical shift data of NeuAc alpha (2----3)Gal beta (1----4)Glc and NeuAc alpha (2----3)Gla beta-(1----4)GlcNAc and their respective NeuAc alpha (2----6) isomers established distinct and different conformations of the sialic acid residue, depending on the type of anomeric linkage [alpha-(2----3) vs. alpha (2----6)]. Interactions between the NeuAc residue and the Glc or GlcNAc residue are particularly strong in the case of the alpha (2----6) isomers. Similar effects are observed for the larger oligosaccharides [II3(NeuAc)2Lac and IV6NeuAcLcOse4] and even in intact glycoproteins and polysaccharides. It is proposed that the NeuAc alpha (2----3) isomers assume an extended conformation with the sialic residue at the end (terminal) of the oligosaccharide chain or branch. The NeuAc alpha (2----6) isomers are assumed to be folded back toward the inner core sugar residues.     

Different fine binding specificities of monoclonal antibodies to disialosylganglioside GD2

The fine structural specificities of six monoclonal antibodies (MAbs) to ganglioside GD2, GalNAc beta 1----4(NeuAc alpha 2----8NeuAc alpha 2----3)Gal beta 1----4Glc-Cer, were studied. The binding specificities of these MAbs were found to differ from each other by virtue of their binding to structurally related authentic standard glycolipids as revealed by three different assay systems, including enzyme immunostaining on thin-layer chromatography, enzyme-linked immunosorbent assay, and immune adherence inhibition assay. The MAbs examined could be divided into three binding types. MAbs A1-201, A1-410, and A1-425 bound specifically to ganglioside GD2 and none of the other gangliosides tested. Two other MAbs (A1-245 and A1-267) reacted not only with GD2, but also with several other gangliosides having the sequence NeuAc alpha 2----8NeuAc alpha 2----3Gal (GD3, GD1b, GT1a, GT1b, and GQ1b). The reactivities with these gangliosides varied to some degree. In addition, these MAbs were found to react with both GD3(NeuAc-NeuAc) and GD3(NeuGc-NeuAc), but not with GD3(NeuAc-NeuGc) or GD3(NeuGc-NeuGc). The last MAb (A1-287) also reacted with several other gangliosides but with lower avidity than A1-245 and A1-267. These findings suggest that each MAb to ganglioside GD2 may have an individual binding specificity and avidity. These MAbs represent potentially useful reagents for analyzing the function of GD2 on cell surface membranes, and provide a system for precisely studying the interactions between an anti-ganglioside antibody and the binding epitope of the antigenic determinant.